Light Quality Modulates the Antioxidant Properties of “Microtom” Fruits: A Pilot Study Testing the Radioprotective Effect on Human Cells

1. Introduction

Tomatoes (Solanum lycopersicum L.), archetypal example of the Mediterranean diet where they are widely consumed as vegetables, play an essential role in human nutrition due to their well-recognized health benefits [1]. They are rich in vitamins, minerals, fibers, proteins, essential amino acids, monounsaturated fatty acids, carotenoids, and phytosterols [2,3,4]. Moreover, tomatoes are an excellent source of bioactive compounds with natural antioxidant properties, such as carotenoids (β-carotenoids and lycopene), ascorbic acid (vitamin C), tocopherol (vitamin E), and are highly abundant in secondary metabolites, like phenolic compounds (quercetin, kaempferol, naringenin, and lutein, as well as caffeic, ferulic, and chlorogenic acids). These compounds are valuable in preventing human pathologies, such as cardiovascular disease (CVD) and neurodegenerative impairment, also exerting an anti-inflammatory action that may reduce cancer risk [5,6,7]. Therefore, the regular consumption of tomatoes may help alleviate symptoms or prevent the onset of several diseases, particularly those feeding on chronic inflammatory conditions [8]. At the cellular level, such beneficial health properties are likely due to a concentration-dependent protection that these molecules may afford to normal cells [9] by scavenging free radicals and neutralizing Reactive Oxygen Species (ROS), suppressing apoptosis and metal chelation, aiding DNA damage repair, and modulating enzyme activities, cytokine expression and signal transduction pathways [10,11,12].

Among tomato cultivars, “Microtom” presents peculiar traits: parthenocarpy[13,14], dwarf size allowing growth at high plant density in small indoor systems (up to 1357 plants m⁻²), and a short life cycle (70–90 days from sowing to fruit ripening)[14]. Moreover, its intrinsic richness in bioactive compounds can be potentially enhanced through targeted light manipulation [15,16]. In fact, although Microtom already boasts high levels of antioxidants and other health-promoting phytochemicals, recent studies under controlled environments have demonstrated that the application of specific light-quality regimes, using the Light Emitting Diode (LED) technology, can significantly boost the synthesis and accumulation of these molecules. This makes Microtom an ideal candidate for producing nutrient-rich fruits and specialized extracts tailored for functional foods and nutraceuticals [17,18].

Recent studies have gone a step further, leveraging Microtom’s unique characteristics to develop a kind of ‘super-extracts’. For instance, research by Vitale et al. [15] and Hay Mele et al. [19] has shown that by optimizing light quality during cultivation, it is possible to obtain Microtom extracts with exceptionally high concentrations of antioxidants and phenolic compounds. These ‘super-extracts’ exhibit enhanced biological activity, making them highly attractive for several applications. Similarly, Paolillo et al. [20] have implemented comparable strategies in Brassica species, underscoring the versatility and scalability of this approach. Notably, the methodology is not limited to tomatoes or brassica: Costanzo et al. [21] have successfully translated these techniques to other crops such as mandarin, suggesting broad applicability across diverse plant systems.

The implications of these innovations are far-reaching. The production of highly enriched plant extracts opens avenues for research and development in multiple sectors. Besides dietary supplementation, pharmaceutical and cosmetics, Microtom-enriched extracts may lend themselves to salutary applications in medicine, providing a natural radioprotection approach to mitigate cellular damage caused by ionizing radiation (IR) in patients undergoing cancer radiotherapy (RT). RT has arguably proven to be a highly successful treatment modality, either alone or in combination with chemotherapy, surgery, immunotherapy and other novel anti-cancer strategies [22,23,24,25,26,27,28,29]. However, RT very often entails irradiation of healthy tissues and organs at risk, especially in the case of deep-seated solid tumours. The radiosensitivity of these tissues is the factor that limits the maximum dose that can be safely administered to achieve successful tumour control without increasing the occurrence of acute adverse reactions, such as skin erythema, or late complications, such as fibrosis or RT-induced secondary cancers [30]. Consequently, there is a strong need for radioprotectors to effectively counteract such deleterious effects. Plant-derived polyphenols have attracted attention due to their natural origin and expected lower intrinsic cytotoxicity [31]. To act as viable radiomitigators, these compounds must disrupt the molecular pathways responsible for the generation of highly reactive species following IR exposure. Via sustained levels of oxidative stress, these mechanisms can perpetuate the initial damage inflicted to DNA by radiation, leading to long-term genetic instability in the progeny of sublethally damaged normal cells/tissues [32]. ROS and reactive nitrogen species reinforce IR-induced geno- and cyto-toxicity, thereby increasing the risk of sequalae [33]. By counteracting oxidative stress, it is possible to prevent structural and functional alterations of proteins and lipids, too [33]. Persistent oxidative stress, e.g., in the form of elevated ROS levels, may impair lipid metabolism, whose dysregulation is implicated in the alteration of the heterogeneous tumor microenvironment by promoting cancer cell proliferation and perturbing the crosstalk between the redox system and lipid metabolic reprogramming [34].

In the rapidly evolving field of nutraceuticals, natural compounds may therefore combine their ability of protecting normal tissue during curative cancer RT, while offering additional benefits when integrated into the diet of oncological patients or the general population to reduce the risk of cancer and CVD. This work evaluated whether different light-quality regimes, namely fluorescent light (FL) and red-blue (RB), under which tomato fruits were grown, may strengthen the antioxidant profile of Microtom fruits and increase the concentration of polyphenols in tomato extracts. We also investigated in vitro if such polyphenolic extracts may confer radioprotection to IR-exposed normal human cell lines. To this end, we used pre-clinically relevant radiobiological assays to quantify the putative extract-induced reduction in DNA damage and oxidative stress levels following doses of high-energy photons as employed in RT. Preliminary tests conducted on an immortalized human skin cell line showed a significant reduction in radiation-induced micronuclei (MN) frequency when treated with extracts from RB-grown tomatoes at a specific concentration. Encouraged by these results, we extended our analysis to both light conditions, testing two concentrations for each condition on a breast epithelial cell line widely considered as a reliable in-vitro model for normal-tissue radioresponse in breast cancer (BC) [35,36].

2. Materials and Methods

2.1. Plant Growth Conditions and Experimental Design

Dry seeds of Solanum lycopersicum L. cultivar “Microtom”, provided by Holland Online Vof (Amsterdam, The Netherlands), from the same batch, were sown in 1.2 l pots containing potting soil composed of 86% peat, 9% sand, 3% quartz sand, 2% perlite. These pots were kept in darkness until germination. Fifteen days after sowing (DAS), once seedlings had emerged, they were transferred to a growth chamber and exposed to two different light-quality regimes, with five pots per light treatment: fluorescent white (FL) and red-blue (RB). These light regimes were chosen based on previous studies demonstrating enhanced photosynthesis and fruit quality in this cultivar [16,37]. The FL regime was achieved by means of fluorescent tubes (Lumilux L36W/640 and L36W/830, Osram, München, Germany), while RB consisted of a combination of red (R: 60%) and blue (B: 40%) light-emitting diode (LED) arrangements (LedMarket Ltd., Plovdiv, Bulgaria). The spectral profiles of the light treatments (Figure 1) were measured using a SR- 3000A spectroradiometer with 10 nm resolution (Macam Photometrics Ltd., Livingston, Scotland, UK). The Photosynthetic photon flux density in the growth chamber was maintained at 360 μmol photons m^-2 s^-1 at canopy level for both light conditions, with a 14-h photoperiod. Environmental conditions were controlled at 60-70% relative humidity and day/night air temperature of 25/20 °C ± 2 °C. Plants were watered weekly to field capacity to compensate for evapotranspiration losses and fertilized weekly with half-strength Hoagland's solution. Plant growth was monitored up to 120 DAS until fruit ripening. After being collected, fruits were powdered with liquid nitrogen by a mortar and a pestle, and stored at -80 °C in the Ecology Laboratory (University of Naples Federico II), until further biochemical analyses and extract preparation for radiobiological in-vitro tests on cell cultures.

2.2. Determination of Total Polyphenol, Flavonoid and Anthocyanin Content

Samples (0.200 g) were extracted in methanol. The extracts were kept for 1 h at 4 °C and then centrifuged at 11,000 rpm for 5 min.

To assess the total polyphenol content, methanolic extracts were mixed with 1:1 (v:v) 10% Folin–Ciocâlteu reagent and shaken. After 3 min, the mixture was complemented with 700 mM Na₂CO₃ solution in the ratio 5:1 (v:v). Samples were incubated for 2 h in the dark. The absorbance was read at 765 nm with a spectrophotometer (UV-VIS Cary 100, Agilent Technologies, Palo Alto, CA, USA) and the polyphenol concentration was estimated calculated using a gallic acid standard curve and expressed as milligrams of gallic acid equivalents per gram of fresh weight (mg GAE g^-1 FW)[38].

Total flavonoid content was estimated following the procedures by Moulehi et al.[39] and Sun et al. [40]. The methanolic extracts were mixed with 5% NaNO₂ solution and, after 6 min, with 10% AlCl₃ and 1 M NaOH solutions. The volume of the mixture was adjusted with distilled water, and the absorbance was read at 510 nm. Total flavonoid content was calculated with a standard catechin (CE) curve and expressed as milligrams of catechin equivalent per gram of fresh weight (mg CE g^-1 FW).

The anthocyanin concentration was quantified as reported by Mancinelli et al.[41] and Chung et al. [42]. Samples (0.250 g) were extracted in the dark for 24 h using acidified methanol (1% HCl) at 4 °C. Once centrifuged, the absorbance of supernatants was read at 530 and 657 nm. The anthocyanin content was quantified by the following equation: (µmoL g^-1) = [(A₅₃₀ – 0.33 x A₆₅₇)/31.6] x [volume (ml)/weight (g)], where the extinction coefficient is 31.6 M^-1 cm^-1.

2.3. Determination of Total Carotenoid, Lycopene and Ascorbic Acid Content

Total carotenoid content was quantified according to Lichtenthaler [43]. Samples (0.100 g) were extracted with cold 100% acetone and centrifuged (Labofuge GL, Heraeus Sepatech, Hanau, Germany) at 5,000 rpm for 5 min. Absorbance was measured with a spectrophotometer (Cary 100 UV-VIS, Agilent Technologies, Santa Clara, CA, USA) at 470, 645 and 662 nm wavelengths, and carotenoid concentration was expressed as milligrams per gram of fresh weight (mg g^-1 FW).

The quantification of lycopene concentration was performed as reported by Fish et al.[44] and Davis et al. [45] with slight modifications. Samples (0.500 g) were mixed in a beaker with hexane: ethanol: 0.05% BHT in acetone (v:v:v, 2:1:1), preventively preparing the solution of butylated hydroxytoluene (BHT) in acetone at the final concentration of 0.05% (w:v). The mixture-containing beaker was then placed on a stirring plate for ~15 min at room temperature. After shaking, deionized water was added to each sample, and the shaking step was repeated for 15 min. The sample was then left at room temperature for 5 min to allow phase separation. The upper hexane layer was transferred into a test tube, and the absorbance of this fraction was read in a 1-cm path length quartz cuvette at 503 nm. The extinction coefficient of lycopene in hexane (17.2 x 104 M^-1 cm^-1) and the lycopene molecular weight (536.9 g mol^-1) were used to convert the absorbance into lycopene concentration expressed as milligrams per gram of fresh weight (mg g^-1 FW).

The ascorbic acid (AsA) concentration was determined using the AsA Assay Kit (MAK074, Sigma-Aldrich, St. Louis, MO, USA), following the procedure reported by Costanzo et al. [46]. The sample (10 mg) was homogenized in 4 volumes of cold AsA buffer and then centrifuged at 13,000 rpm for 10 min at 4 °C. The volume of the liquid fraction was adjusted with the AsA assay buffer and mixed with the kit reagents to start the colorimetric coupled enzyme reaction. The colored product developed from the reaction is proportional to the amount of AsA contained in the sample. The absorbance was read at 570 nm and the AsA concentration was calculated using a standard curve and expressed in ng µl^-1.

2.4. Antioxidant Capacity (FRAP assay) and Free Radical Scavenging Activity (DPPH Assay)

The antioxidant capacity was assessed through the Ferric Reducing Antioxidant Power (FRAP) assay, according to George et al. [47] and modified by Costanzo et al. [46]. Samples (0.250 g) were extracted in methanol: water solution (60:40, v:v) for 1 h and centrifuged for 15 min at 14,000 rpm at 4 °C. The extracts were mixed with the FRAP reagents and incubated in the dark for 1 h. After the reaction, the absorbance was read at 593 nm. The antioxidant capacity was determined using a Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid) standard curve and quantified as µmol Trolox equivalents per gram of fresh weight (µmol TE g⁻¹ FW).

The free radical scavenging activity of tomato fruit extracts was evaluated using the 1,1-diphenyl-2-picrylhydrazyl (DPPH) assay, according to Dudonné et al. [48] and modified by Costanzo et al. [46]. An aliquot (67 μl) of methanolic extract was added to 2 ml of 6 × 10^-5 M DPPH methanolic solution. The mixture was stirred and incubated at 37 °C for 20 min. The absorbance of the yielded sample was read at 515 nm and converted to DPPH radical inhibition percentage using the following equation:

$$\mathrm{I}\mathrm{n}\mathrm{h}\mathrm{i}\mathrm{b}\mathrm{i}\mathrm{t}\mathrm{h}\mathrm{i}\mathrm{o}\mathrm{n}\left(\mathrm{\%}\right)={\displaystyle \frac{{\mathrm{A}\mathrm{b}}_{\mathrm{b}\mathrm{l}\mathrm{a}\mathrm{n}\mathrm{k}}-{\mathrm{A}\mathrm{b}}_{\mathrm{s}\mathrm{a}\mathrm{m}\mathrm{p}\mathrm{l}\mathrm{e}}}{{\mathrm{A}\mathrm{b}}_{\mathrm{b}\mathrm{l}\mathrm{a}\mathrm{n}\mathrm{k}}}}\times 100$$

(1)

where Ab_blank is the absorbance of the blank, and Ab_sample is the absorbance of the tested methanolic extract. Trolox was used as the positive control.

2.5. Cell Cultures

Two cell lines were used in this work. They were grown and serially sub-cultivated according to conventional cell culture techniques. The spontaneously immortalized HaCaT cell line, derived from adult human epidermal cells and retaining the capacity to proliferate and differentiate in a manner similar to basal keratinocytes in vivo, was purchased from the Istituto Zooprofilattico Sperimentale della Lombardia e dell’Emilia Romagna (IZS-LER), Brescia, Italy. HaCaT cells were grown in DMEM (Dulbecco's Modified Eagle Medium), supplemented with 10% fetal bovine serum, 1% penicillin/streptomycin, 1% L-glutamine, and 1% sodium pyruvate (Gibco, ThermoFisher Scientific, US). Non-tumorigenic breast epithelial MCF-10A cells were kindly donated to the Radiation Biophysics Laboratory, University of Naples Federico II, by Dr P. Chaudhary, Queen’s University Belfast, UK. As described in detail by Debnath et al. [49], two DMEM/F12-based culture media (Gibco, ThermoFisher Scientific, US) are required for maintenance of these cells: one formulation is intended for optimal growth, and is enriched with 5% horse serum, endothelial growth factor (20 ng ml^-1), hydrocortisone (0.5 µg ml^-1), insulin (10 µg ml^-1), and cholera toxin (100 ng ml^-1), all from Sigma-Aldrich, Merck, Germany; the other, devoid of all supplements but rich in horse serum (20%), is used for routine passaging, i.e. for trypsin quenching and dilutions for cell counting prior to plating. Both cell lines were grown in standard tissue T75 culture flasks placed at 37 °C in a humidified atmosphere with 5% CO₂.

2.6. Extract Treatment of Cell Cultures

To evaluate the putative radioprotective properties of tomato fruits grown under FL and RB light regimes, the phenolic extracts were preventively dried at a temperature of 50°C for 30 min in a Rotavapor RII (BÜCHI Labortechnik AG ®, Flawil, Switzerland). The dry material was weighed and solubilized in methanol to reach the concentration of 1000 mg ml^-1 (stock solution). The phenolic extracts were then tested at two final concentrations, 100 and 200 mg ml^-1, by adding them to the medium of exponentially growing cells in either slide flasks (Nunclon, ThermoFisher Scientific, US, 9 cm^-2 growth surface) for DNA damage evaluation by the cytokinesis-block micronucleus (CBMN) assay or in the T25 tissue culture flasks for the ROS assay. Cultures were thus incubated for 24 h and then exposed to radiation in the presence of the extracts. Immediately after irradiation, cells were rinsed thoroughly by phosphate-buffered saline (PBS) solution and subjected to the assay procedure. Cultures irradiated in the absence of the extracts served as controls.

2.7. Cell Exposure to Clinical Photons

Irradiations of cell cultures prepared as above described were performed at Istituto Nazionale Tumori, IRCCS Fondazione G. Pascale, Naples, Italy, by a Synergy Agility LINAC (Elekta, Sweden). Such an accelerator is conceived to deliver RT doses for treatments of oncological patients; therefore, routine quality control checks are performed to make sure the dose delivered adheres to the prescribed one as computed by a Treatment Planning System (TPS). Three-dimensional conformal radiation therapy (3D-CRT) treatment plans were designed specifically for these experiments by means of a dedicated software (Monaco v5.11.03 TPS, Elekta, Sweden). Flasks were imaged by a Computed Treatment (CT) scan in the exact position they were treated.

By means of the CT-image based TPS, the volume to be treated was thus contoured and the calculation of the prescribed radiation dose to be administered was performed. To achieve a homogeneous dose distribution within the irradiated field, the geometric setup consisted of two opposing fields at 0° and 180°. Specifically, two 6-MeV squared photon beams covering a surface of 20 x 20 cm² were used to deliver doses of 0.5, 2 and 4 Gy to the cells. To ensure adequate build-up during dose delivery by the LINAC rotating gantry, flasks were placed between layers of solid-water slabs of appropriate thickness, namely 2.8 cm for the upper slab and 5 cm for the lower slab. The associated dose uncertainty was estimated to be less than 3%, in line with standard tolerances routinely adopted in RT.

2.8. Measurement of Radiation-Induced DNA Damage

The well-established CBMN genotoxicity assay [50] was performed to evaluate the putative radioprotective effect exerted on DNA by the tomato extracts from plants grown under FL and RB light-quality regimes. In the CBMN assay, DNA damage manifests itself in the form of round portions of DNA, called micronuclei (MN), originated from lagging chromosomes or acentric chromosome fragments at anaphase [51,52] that fail to be incorporated into either of the two daughter nuclei. Based on this, because MN can be associated to the daughter nuclei before cytokinesis ends the telophase stage, they must be scored in binucleated (BN) cells. The amount of IR-induced damage is quantified by the presence of those MN that meet well-defined morphological criteria as described by Fenech et al. [50]. The actin-destroying agent Cytochalasin B (CytB), which inhibits cleavage of the cytoplasmic furrow during cytokinesis, was used for arresting cells at the BN stage. Measurement of the MN frequency in BN HaCaT and MCF-10A cells allowed the evaluation of eventual mitigation of radiation-induced genotoxicity following treatment with the tomato extracts.

2.8.1. Micronucleus Frequency Measurement

After photon irradiation (0, 0.5, 2 and 4 Gy), cells were treated for 24 h with 2.0 μg ml^-1 of CytB. These were the optimal treatment duration and CytB concentration as determined by preliminary tests as to yield the highest proportion of BN cells compatibly with the lowest CytB-induced baseline genotoxicity. Subsequently, the CytB-containing medium was removed, cells were rinsed in PBS and fixed for 20 min at -20°C with freshly prepared 4:1 Carnoy's solution (methanol: acetic acid) that had been pre-cooled at -20°C for 30 min. After removal of the fixative solution, slides were air-dried for 24 h and then stained with 14 μl of ProLong Gold DAPI in antifade (ThermoFisher Scientific, US). Controls samples were subjected to the same procedure. The analysis was then manually performed with an Axioplan 2 epi-fluorescence imaging microscope (Carl Zeiss AG, Germany), using a 40X magnification objective. MN frequency was determined in BN cells according to the formula:

$$f={\displaystyle \frac{{\mathrm{M}\mathrm{N}}_1+{(2\times \mathrm{M}\mathrm{N}}_2)+{(3\times \mathrm{M}\mathrm{N}}_3)+{(4\times \mathrm{M}\mathrm{N}}_4)}{\mathrm{B}\mathrm{N}}}$$

(2)

where MN_n is the number of BN cells with n MN, and BN is the total number of BN cells scored. Between 500 and 1,500 BN cells were scored for each sample, depending on the radiation dose, for statistical robustness.

2.9. Determination of Radiation-Induced Oxidative Stress

The level of oxidative stress was determined in MCF-10A breast epithelial cells to evaluate the antioxidant action exerted by the tomato extracts from plants grown under FL and RB light-quality regimes. Cells were irradiated with the same doses employed for the CBMN assay, i.e., 0, 0.5, 2 and 4 Gy. Oxidative stress was measured by means of a flow cytometer-based assay (Invitrogen Attune NxT Flow, ThermoFisher Scientific, US) using a commercially available kit composed of the fluorogenic CellROX™ Green Reagent for measurement of cellular oxidative stress in live cells, and the SYTOX™ AADvanced™ Dead Cell Stain Kit (both from ThermoFisher Scientific) to discriminate live cells against the dead cell population. Upon oxidation by ROS and subsequent binding to DNA, the cell-permeant CellROX dye, weakly fluorescent while in a reduced state, emits bright green, fluorescent light, with absorption/emission maxima of 485/520 nm.

2.9.1. ROS Level Measurement

Cells were treated 1 h after irradiation. Firstly, they were harvested by trypsinization and centrifuged (200 g for 5 min). The pellet was resuspended in medium using Eppendorf tubes to a concentration of about 2·10⁵ cells ml^-1. The staining procedure by the CellROX green (250mM) was carried out for 30 min in the incubator at 37°C, using 0.5 ml of cell suspension containing a final number of 1·10⁵ cells in accordance with the manufacturer’s guidelines; afterwards, the Sytox AADvanced stain (1µl per 1ml of cell suspension) was added for 5 min at room temperature. Flow cytometry analysis followed immediately. “Blank” samples, i.e. without fluorescent probes, were used to set the mean cellular autofluorescence level as to avoid overestimating the presence of intracellular ROS and, hence, to position the quadrants to discriminate between alive non-stressed cells, alive stressed cells, false double positives and dead cells. Cells not treated with the tomato extracts were used as controls and underwent the same experimental procedure.

2.9. Statistical Analysis

Statistical analysis and data representation were performed using Sigma Plot 12.0 (Jandel Scientific, San Rafael, CA, USA). Concerning biochemical investigations, statistically significant differences between FL and RB samples were checked by T-test, based on a significance level of p < 0.05. All data were expressed as means ± SE (n = 8). As regards the radioprotective effect of tomato extracts, within each radiation dose (0, 0.5, 2 and 4 Gy) and for each extract concentration (100 and 200 mg ml^-1), FL and RB-treated cell lines were compared to controls by a T-test based on a significance level of p<0.05. The normal distribution of data was verified by Shapiro–Wilk test. Error on the frequency f of MN and on that of ROS-positive cells was assumed to follow a binomial distribution per each individual experiment according to formula 3:

$$Error= \sqrt{{\displaystyle \frac{f\times |1-f|}{n}}}$$

(3)

For both CBMN and ROS assays, results were then plotted as the weighted means (formula 4) with relative SE (formula 5) from at least three separate experiments per each cell line, extract concentration, light-regime quality, and radiation dose, the individual weights w_i being the inverse of the squared SE:

$$Weighted mean= {\displaystyle \frac{\sum _{i=1}^3{w}_i f_i}{\sum _{i=1}^3{w}_i}}$$

(4)

$$Weight error=\sqrt{{\displaystyle \frac{1}{\sum _{i=1}^3{w}_i}}}$$

(5)

3. Results

3.1. Total Phenolic Compounds

Different light-quality regimes significantly influenced the total content of polyphenols, flavonoids, and anthocyanins in tomato fruits (Figure 2).

The greatest amount of polyphenols was found in tomato fruits treated with RB light regime (26.3 ± 0.3 mg GAE g⁻¹ FW) compared to FL (23.9 ± 0.3 mg GAE g⁻¹ FW) (Figure 2A). Total flavonoid content varied significantly among RB and FL light regimes, showing higher concentration in RB tomatoes (78 ± 7 mg GAE g⁻¹ FW) than in FL (51 ± 4 mg GAE g⁻¹ FW) (Figure 2B). Conversely, total anthocyanin content increased in tomatoes treated with RB light regime (11.7 ± 0.6 μmol g^-1 FW) compared to FL (10.2 ± 0.4 μmol g^-1 FW) (Figure 2C).

3.2. Total Carotenoids, Lycopene and Ascorbic Acid

Total carotenoid content varied among different light regimes. RB fruits exhibited a higher concentration of carotenoids (0.110 ± 0.001 µg g^-1 FW) than those derived from FL (0.084 ± 0.001 µg g^-1FW) (Figure 3A). Total lycopene content varied among different light regimes too. RB fruits exhibited a higher concentration of lycopene (30.4 ± 0.05 µg g^-1 FW) than those from FL (26.1 ± 0.1 µg g^-1 FW) (Figure 3B). Finally, light quality significantly affected the ascorbic acid (AsA) content (Figure 3C). More specifically, FL regime (1.95 ± 0.17 ng μl^-1) showed a higher amount of ascorbic acid compared to RB light (1.40 ± 0.09 ng μl^-1).

3.3. Sample Antioxidant Activity

Total antioxidant capacity evaluated by the FRAP assay significantly varied between RB and FL light regimes (Figure 4). Similarly to the case of total polyphenols, the highest value was observed in tomatoes treated with RB light (4.9 ± 0.3 μmol Trolox eq^-1 FW) compared to FL (4.1 ± 0.1 μmol Trolox eq g^-1 FW). The DPPH radical scavenging assay also confirmed that the antioxidant activity changed according to the different light-regime treatments. More specifically, the highest value was found in tomatoes treated with the RB regime (45.3% of inhibition) compared to FL (38.8% of inhibition).

3.4. Radiation-Induced Genotoxicity

A preliminary series of experiments was carried out in the human keratinocyte HaCaT cell line with the aim to pre-screen a possible radioprotective action by the tomato extracts biochemically characterized as described above. To this end, the anti-genotoxic activity of the RB extract (200 µg ml⁻¹) was tested using the CBMN assay, as shown in Figure 5. Data clearly show that the extract did not increase the baseline MN frequency in unirradiated HaCaT cells (0 Gy), indicating that the RB extract does not cause genotoxicity per se. At all radiation doses, the presence of the RB-grown tomato extract significantly reduced MN frequency compared to cells irradiated without extract. At the lowest doses used, i.e., 2 and 0.5 Gy, RB-treated cells showed a marked reduction in MN frequency, between 30% and 25%, respectively. Although the protective effect appeared to diminish at the highest dose of 4 Gy, the extract-associated reduction in MN frequency remained statistically significant (p<0.05).

Investigation into whether the radioprotective effect observed for the RB-derived extract at the 200 mg ml^-1 concentration in the HaCaT cell line could also be detected at lower concentrations, and whether such radioprotection could be influenced by plant growth light regimes, was extended to the breast epithelial cell line MCF-10A, due to its pre-clinical relevance for healthy tissue radioprotection in breast cancer (BC) patients. Modulation of cellular radioresponse by the two concentrations of the extracts derived from fruits grown under FL- and RB-light regimes is presented in Figure 6. Similar to what had been found in HaCaT cells, the extract, irrespective of the light regimes, did not affect the baseline MN frequency in MCF-10A cells. Except for 100 mg ml^-1 FL-derived extracts following exposure to 0.5 and 2 Gy of clinical photons (Figure 6A), both extract concentrations and light regimes demonstrated a significant radioprotective effect on cellular DNA compared to extract-untreated irradiated cells (Figure 6). Interestingly, the RB light regime resulted in concentration-dependent radioprotection, which exceeded the magnitude of the FL extract protection at each radiation dose. In detail, at the highest concentration (200 µg ml^-1) under RB light, the reduction in mean MN frequency ranged from 30 to 40% relative to untreated controls, consistently outdoing the effect observed at 100 mg ml^-1 (Figure 6). For instance, after 0.5 Gy, a dose close to those affecting the healthy tissue in RT, 100 µg ml^-1 of RB extract decreased MN frequency by 20% compared to the untreated sample, while at the same radiation dose, a concentration of 200 µg ml^-1 achieved a reduction of 39% (Figure 6A vs. Figure 6B). Notably, at the highest tested radiation dose (4 Gy), the MCF-10A cells did not exhibit the decrease in radioprotection measured in the HaCaT cell line (Figure 5).

Figure 7 portrays examples of scored MN in 0.5 Gy-irradiated BN MCF-10A cells treated with 200 µg ml-1 RB and FL extracts: note the presence of multiple MN in extract-untreated BN cells (Figure 7D) and the extract-mediated attenuation of damage in RB- and FL-treated ones as shown Figure 7B,C, respectively.

3.5. Oxidative Stress Levels

The elevation of ROS-associated oxidative stress following exposure of MCF-10A cells to clinical photons was evaluated by the flow cytometry-based CellROX™ assay. Both FL and RB extracts were tested at a concentration of 200 µg ml^-1 since this was found by the CBMN assay to provide the greatest reduction in radiation-induced DNA damage (Figure 6). As shown in Figure 8, radiation exposure led to a marked dose-dependent increase in the baseline level of intracellular oxidative stress in extract-untreated samples as measured 1h post irradiation. Importantly, however, tomato extracts were able to significantly reduce such stress levels in both unirradiated controls and irradiated cells. This antioxidant effect was observed for both light regimes although RB generally demonstrated greater efficacy, particularly at higher doses (i.e., at 2 and 4 Gy). Moreover, treatment with RB nearly restored ROS levels in cells exposed to 0.5 and 2 Gy to those measured in unirradiated controls (0 Gy). Specifically, the RB extract reduced ROS levels by approximately 43% at 0.5 Gy and 55% at 2 Gy compared to irradiated cells without treatment.

4. Discussion

This study demonstrates that different light-quality regimes can influence the concentration of bioactive molecules and the antioxidant capacity of Solanum lycopersicum L. cultivar “Microtom” tomato fruits, confirming that modulation of the light spectrum is a valuable tool for enhancing fruit nutritional and functional properties [53]. Notably, for the first time, we provide in vitro evidence that extracts from these tomatoes exhibit pronounced radioprotective activity in irradiated non-cancer cells. This may have important implications for the welfare of cancer patients undergoing radiotherapy (RT). Mitigation of radiation-induced DNA damage and intracellular oxidative stress was proven by a significant reduction in the frequency of micronuclei (MN) and by lowering of the ROS levels by 100 and 200 µg ml^-1 of extracts from fruits grown under fluorescence (FL) and red-blue (RB) light regimes.

The total antioxidant capacity of tomato fruits primarily arises from ascorbic acid (AsA), carotenoids, and polyphenols. FL light, for example, enhances AsA content in tomato fruits due to its broader spectrum, including far-red and UV wavelengths that can activate additional photoreceptors and signaling pathways involved in ascorbate biosynthesis. As demonstrated by Hay Mele et al. [19], tomatoes grown under fluorescent or full-spectrum light exhibited significantly higher AsA levels than those cultivated under RB light, underscoring the importance of specific wavelength ranges in modulating antioxidant metabolism. While exposure to blue light is known to increase AsA content in detached tomato fruits compared to white, red, or green light [54,55,56,57,58,59,60,61], our study found that the enhanced antioxidant activity in fruits exposed to RB light was not due to increased AsA. Instead, it was attributed to elevated carotenoids and phenolic compounds [19,38,55]. This aligns with previous observations in the same cultivar, where an increase in total antioxidant capacity under RB regime was linked to a concomitant rise in polyphenols, including flavonoids, anthocyanins, and lycopene among carotenoids, particularly stimulated by red and blue wavelengths [19,38,54,62]. These bioactive compounds enhance the nutritional value of fruits and vegetables and contribute to multiple health benefits, mainly through their antioxidant and anti-inflammatory roles. In tomatoes, the synthesis of polyphenols is strongly modulated by blue and red light, leading to increased antioxidant capacity and ROS scavenging potential. A recent study has demonstrated that tomatoes grown under RB light produce fruits richer in quercitin and derivates, like quercitin rutinoside, quercitin 3-O-Galactoside, and quercitin acetylhexoside [19,38]. These compounds are known for their free radical scavenging activity, modulation of pro-inflammatory cytokines, and inhibition of cancer cell proliferation. Notably, rutin and quercitin rutinoside have been associated with protective effects against cardiovascular diseases [19,63,64]. Additionally, RB tomatoes extracts enriched in chlorogenic acid and caffeoylquinic acid derivates, apigenin and apigenin-8-c-Glucoside, recognized for their anticancer and anti-inflammatory properties [65,66,67,68]. Therefore, the regular intake in daily diet of such antioxidant compounds through fruits and vegetables can counteract ROS, promote human health [69,70], exert antiproliferative and pro-oxidant effects on tumor cells [71], while providing anti-inflammatory activity in normal cells [72].

Despite the encouraging properties against oxidative stress-induced genotoxicity [20], scant data exists on the protective effects of tomato fruit-derived compounds on radiation-induced cytogenotoxicity in the therapeutic context of counteracting radiotoxicity of healthy tissues [73,74]. To our knowledge, this is the first report linking light-dependent metabolic modulation in tomatoes to radioprotective efficacy in normal human cells.The chosen radiobiological endpoints (MN induction and ROS levels) hold valuable pre-clinical relevance. The presence of MN in irradiated cells reflect chromosomal damage and mis-segregation that, if sublethal to the surviving proliferating cell, can be transmitted through cell division, compromising genomic stability and potentially inactivating the newly discovered cGAS-Sting pathway that stimulates systemic immune response [75,76,77]. Similarly, the perpetuation of oxidative stress is a known risk factor for RT-related secondary cancers [60,78].

The varying degrees of radioprotection observed in our study, directly influenced by the modulation of light regimes, indicate that extracts from plants grown under an RB light regime are more effective than those from FL conditions in counteracting radiation-induced genototoxicity (Figure 5 and Figure 6) and oxidative stress (Figure 8). The enhanced efficacy of RB-grown tomatoes appears to correlate to a marked increase in key antioxidant compounds, especially polyphenols. This enrichment is not merely a quantitative change; RB lighting triggers specific photoreceptors and transcriptional programs that enhance the biosynthesis of these molecules, effectively ‘priming’ the fruit to produce higher levels of defensive secondary metabolites, well known for their ability to scavenge radiation-induced ROS, stabilize cellular structures, and mitigate DNA damage in irradiated cells.

In this context, natural compounds can reduce the adverse effects of RT and improve the tolerance to radiation of healthy cells [79]. Antioxidant compounds, as secondary metabolites, play a key role in plants, especially as a defense strategy in response to environmental stressors. Among these, light represents one of the most powerful environmental cues modulating plant metabolism. Different wavelengths can activate transcriptional pathways that lead to the synthesis and accumulation of antioxidant molecules [80,81].

In humans, through the ingestion of fruits and vegetables, these compounds act as antioxidants by counteracting ROS and promoting human welfare [82,83]. Therefore, the modulation of light during plant growth does not only influence agricultural traits but directly impacts on the nutraceutical profile of the harvested fruits, ultimately determining their potential health-promoting properties. Food-isolatable nutraceuticals demonstrate antiproliferative and pro-oxidant effects on tumors [84,85], alongside antioxidant and anti-inflammatory activity in normal cells [86]. Indeed, the attractive double-edged potential of pure polyphenols or polyphenol-enriched extracts could be used to widen the therapeutic window in RT [87] .

At least as regards the radioprotective capacity of tomato fruits, preliminary experiments revealed that extracts obtained from tomatoes grown under RB light regimes at a concentration of 200 µg ml⁻¹, significantly reduced radiation-induced DNA damage in human HaCaT keratinocytes (Figure 5). This prompted us to focus our study on the MCF-10A cell line using the extracts from both growth light regimes. Not only did we confirm their DNA-protecting action against radiation-induced chromosomal damage, but we also found a pronounced ability to reduce radiation-elevated ROS levels. Since such results were obtained following exposure to photons as used in RT in the MCF-10A cell line, which is widely regarded as an in-vitro model for normal-tissue reaction in breast cancer (BC) patients in vivo [35,36], they hold pre-clinical importance. Hence, the extract-induced radioresistance supports the possible therapeutic use of such extracts as bioactive compounds capable of mitigating the adverse effects of radiation. The radiosensitivity of healthy tissues and organs is the main dose-limiting factor in curative RT because of late-occurring consequences such fibrosis and chronic inflammation. Indeed, this aspect is even more critical for BC, where mitigation of radiation-associated cardiotoxicity, often fueled by pro-inflammatory and pro-oxidant responses [88] has emerged as a priority for treatment of this malignancy [89].

It is important to note that even under baseline conditions, that is when cell cultures were not exposed to radiation, the extracts protected cells by lowering physiologically oxidative stress level. Moreover, the extent of protection granted by the extracts towards radiation-induced effects depended on the specific light regime the plants from which they derived had been grown. Thus, FL reduced the percentage of intracellular ROS, albeit less efficiently than RB. The difference between the extracts is significant for doses of 0, 2, and 4 Gy, while it is comparable at 0.5 Gy. Examining the data at 0 Gy in Figure 6 A,B, we notice that the frequency of MN from tomato extracts is comparable to that of the control. While one might expect a similar trend for oxidative stress, this was not the case. This apparent discrepancy can be explained by the fact that the mechanisms underlying MN formation are not exclusively driven by oxidative damage. MN originating from acentric chromosomal fragments are typically linked to direct DNA double-strand break mis-repair, whereas those containing whole chromosomes result from mitotic mis-segregation. Therefore, oxidative stress is only one of several factors contributing to MN induction. Additionally, it ought to be considered that the probe used in the oxidative stress assay simultaneously assesses the presence of ROS in both the mitochondria and the nucleus. At a baseline level, nuclear ROS levels may remain unchanged, while mitochondrial ROS could be more responsive to the antioxidant activity of the extracts, as suggested by previous reports on compartmentalized ROS signaling [90]. This compartmentalization might thus account for the observed divergence between MN and ROS data at 0 Gy.

In conclusion, our results hold significant implications in BC RT scenarios because, by reducing late normal-tissue complications, particularly those linked with RT-induced cardiovascular disease CVD), through a toxicity-free nutraceutical approach, the therapeutic window for BC RT may be enlarged. Furthermore, such an approach may be interesting for the radioprotection of manned space exploration crews, who are exposed to a peculiar radiation field and physiological conditions [91,92,93], and share similar serious long-term health hazards with RT patients, such as increased risk of radiation-induced CVD and cancer, which also invoke bioactive countermeasures [94,95].

These considerations collectively highlight the novelty of our results and justifies further studies on the radiomodulatory properties of Solanum lycopersicum L. cultivar “Microtom” extracts from fruits grown under different light regimes.

5. Conclusions

For the first time this study evaluated in vitro whether phenolic extracts of “Microtom” fruits, grown under fluorescent white (FL) and red-blue (RB) light regimes, may provide radioprotection on normal human cell lines. Our findings suggest that light spectrum quality can profoundly shape the biochemical profile of tomato fruits. The tomato extracts, administered at 100 and 200 µg ml^-1, effectively mitigated normal-tissue radiotoxicity in normal breast cells exposed to therapeutic photon beams. Notably, the RB extract at a concentration of 200 µg ml^-1, provided the greatest degree of radioprotection. This is in keeping with our findings that tomatoes grown under RB lighting were richer in polyphenols, flavonoids, carotenoids, and lycopene, thereby resulting in higher antioxidant capacity compared to those grown under FL light. These findings underpin the potential of targeted light regimes as powerful tools to produce healthier nutrient-enriched food and their possible usefulness for breast cancer patients undergoing radiotherapy. Overall, our results may represent a starting point for further research aimed at:

• investigating the molecular pathways by which RB light enhances the synthesis of radioprotective polyphenols in tomato fruits;
• testing whether similar light-induced biochemical profiles can be obtained in other edible plant species;
• developing standardized tomato-derived extracts or formulations to reduce radiotherapy-induced normal tissue toxicity.